Obtaining fatty acid esters from native oils and fats

ABSTRACT

Process and device for obtaining fatty acids or fatty acid esters from native oils and fats by their enzymatic hydrolysis and optional simultaneous esterification with alcohols, especially n- and iso-alcohols Process and device for obtaining fatty acids or fatty acid esters from native oils and fats, in which lipases, as biocatalysts for hydrolysis of oils or fats, are caused to act on a mixture of an oil or fat, water, and optionally an oil- or fat-soluble alcohol to hydrolyze the oil or fat and optionally to form an ester, where the reaction mixture thus formed is placed in an self-discharging centrifuge for separation into a glycerol-containing aqueous phase and an organic phase, the centrifuge is adjusted so that a lipase-enriched intermediate phase collects in the centrifuge between the aqueous that is drained off and the organic phase that is drained off, and the centrifuge is emptied at specified times and the discharged drum contents from the centrifuge are returned to the combined hydrolysis or optionally combined hydrolysis/esterification process.

A process and device for obtaining fatty acids or fatty acid esters fromnative oils and fats by their enzymatic hydrolysis and optionalsimultaneous esterification with alcohols, especially n- andiso-alcohols

The invention concerns a process and a device for obtaining fatty acidsor fatty acid esters from native oils and fats by their enzymatichydrolysis and optional simultaneous esterification using alcohols,especially n- and iso-alcohols.

Enzymatic hydrolysis of oils and fats has indeed been known for a longtime, but has not been able to compete with hydrolysis under pressurebecause of the high enzyme consumption.

The fatty acid esters from native fatty acids contained in fats andoils, and medium-chain (chain length less than 6 carbons) to long-chain(generally up to a 24-carbon chain length) n- and iso-alcohols are ofhigh economic importance in numerous applications, especially in thelubricant field.

It is quite difficult to prepare esters of these alcohols withunsaturated fatty acids, especially oleic acid esters, by classicalchemical routes such as acidic esterification. Enzymatic preparation ofthese esters from fatty acids and alcohols has not been carried out inthe past because of the high enzyme requirement.

The objective of this invention is to provide an economically feasibleenzymatic process for producing fatty acids and fatty acid esters fromnative oils and fats, and a corresponding device.

This objective is realized in the subject matter of the independentclaims. Preferred further embodiments are defined in the subclaims.

The inventors have developed an economically efficient enzymatic processfor hydrolyzing fats or oils, and a corresponding device to accomplishthe process. They have also found that this process and device togetherconstitute an outstanding solution to the previous problems of enzymatichydrolysis and enzymatic production of the esters of interest in oneoperation.

The process of the present invention for the enzymatic preparation offatty acid esters is characterized by the fact that lipases, asbiocatalysts for the hydrolysis of oils or fats and for esterification,are caused to act on a mixture of an oil or fat, water, and a fat- oroil-soluble alcohol, especially n- and/or iso-alcohols. The resultingreaction mixture is transferred to a self-discharging centrifuge inorder to separate the glycerol-containing aqueous phase formed in thecombined hydrolysis-esterification process from the organic phase whichcontains the fatty acid esters. The centrifuge is adjusted so that anintermediate layer enriched in lipase (enzyme) that forms between theaqueous and organic phase collects in the centrifuge drum. Thecentrifuge drum is emptied at specified times and the dischargedcontents are returned to the combined hydrolysis and esterificationprocess. The contents of the drum are also available to be used inanother, separate hydrolysis and esterification process, in which isincorporated into the present process or just made available for a laterprocess.

Without addition of the alcohol, enzymatic hydrolysis occurs withoutsimultaneous esterification, and the hydrolysis makes possible anextremely economical production of free fatty acids and glycerol. Thebasic procedure is otherwise identical.

The above-mentioned intermediate layer is enriched in the plate pack ofself-desludging separators, which are suitable as self-dischargingcentrifuges. However, in principal ribbed inserts or other internalstructures such as blades and the like may be used in an equivalentmanner in place of the plate pack. It is important that the centrifugebe self-discharging, making it possible for the intermediate layer thatcollects in the centrifuge to be discharged from time to time byemptying the centrifuge drum.

The above-mentioned intermediate layer forms during the enzymatic fathydrolysis even when alcohol is not added, along with the separation ofthe two phases that form: the glycerol-containing aqueous phase and theorganic phase that contains the free fatty acids formed in thehydrolysis. The problem of this emulsion-like intermediate layer isknown from “Continuous Uses of Lipases in Fat Hydrolysis,” M. Bühler andChr. Wandrey, Fat Science Technology 89, December 1987, pages 598 to605; “Enzymische Fettspaltung” [Enzymatic Fat Hydrolysis], M. Bühler andChr. Wandrey, Fett Wissenschaft Technologie [Fat Science and Technology]89, No. 4, 1987, pages 156 to 164; and “Oleochemicals by BiochemicalReactions?” M. Bühler and Chr. Wandrey, Fat Science Technology 94, No.3, 1992, pages 82 to 94.

In “Continuous Use of Lipases in Fat Hydrolysis,” oil is hydrolyzedcontinuously in a first stirred reactor. The reaction product, which inaddition to free fatty acids contains water, glycerol, and mono- anddiglycerides, unhydrolyzed oil and enzyme, is transferred to a solidwall plate centrifuge which is adjusted so that the intermediate layerbetween the aqueous glycerol phase and the organic phase is dischargedwith the organic phase. The organic phase containing the intermediatelayer is delivered to a second stirred reactor, to which a freshwater/enzyme mixture is also added. The reaction product from the secondreactor is again transferred to a solid wall plate centrifuge, which inthis case is adjusted so that the intermediate layer is discharged withthe glycerol-containing aqueous phase, so that the free fatty acidsproduced will be discharged without any intermediate emulsion layer. Theaqueous phase is returned to the first reaction, so that the enzymeportion contained in the emulsion intermediate layer is resupplied tothat process. Moreover, as is usually the case with self-dischargingcentrifuges, the solids that deposit on the drum wall are dischargeddiscontinuously when the drum is emptied.

An emulsion-like intermediate layer also forms during the phaseseparation in the hydrolysis carried out according to the presentinvention, with or without simultaneous esterification. Thisintermediate layer contains substantial quantities of enzyme, along withthe organic phase that contains the free fatty acids or their esterswhich are formed, and with the glycerol- containing aqueous phase.However, the objective is accomplished successfully with a centrifugehaving exceptionally high capability, and an efficient and nearlyloss-free recycling of the enzyme is achieved. This makes it possible toperform the fat hydrolysis with a high cleavage rate and theesterification in high yield, at high enzyme concentration andconsequently short reaction time, without significant loss of enzyme.

A self-discharging centrifuge is used, preferably a self-dischargingseparator with a plate pack. The centrifuge is adjusted so thatsignificant amounts of the enzyme-containing interfacial emulsion arenot included in the ester-containing organic phase, nor in the aqueousphase. Instead, this emulsion accumulates in the centrifuge, preferablyin the region of the separation zone in the plate pack of a separator.This is an unusual operating configuration to the extent that generallythe accumulation of large quantities of an intermediate layer in theplate package during liquid/liquid phase separation is specificallyavoided with the known self-discharging separators or self-desludgingseparators. On the contrary, one generally takes care that the smallestpossible amount of this intermediate layer is produced, and it isgenerally discharged with the phase which is not the principal one to berecovered. This is also essentially what was done in the abovepublication, in which the intermediate layer was discharged with theaqueous phase from the second continuously operating solid wallcentrifuge and resupplied to the first reactor. Furthermore, withself-discharging centrifuges, generally the solids that deposit on thedrum wall are discharged discontinuously when the drum is emptied.

For one skilled in the art, adjustment of the centrifuge according tothe present invention means applying the measures known to the art foradjusting the weir and/or adjusting the back pressure at the exit portto insure that both the organic phase and the aqueousglycerol-containing phase discharge as clear and free of emulsion aspossible.

In the process of the present invention, the presence of alcohols doesnot result in either significantly higher enzyme consumption orsubstantially extended reaction time. The recovery of the esters fromthe reaction mixture in a simple manner is also possible.

The alcohols are added in the stoichiometrically required proportion forester formation, but it is advantageous to use an excess of 2% to 100%,preferably 5% to 20%, based on the stoichiometric requirement for thecorresponding oils or fats. An excess of alcohol accelerates the fathydrolysis and unexpectedly causes complete hydrolysis of all glyceridesin a short time.

The glycerol formed in the reaction moves into the water phase becauseof its extremely high solubility in water and very low solubility in thehydrophobic organic phase. Since the medium-chain to long-chain alcoholsare very poorly soluble in water but quite soluble in the organic phase,while water on the other hand is very poorly soluble in the organicphase, these alcohols are converted enzymatically to fatty acid estersaccording to the chemical equilibrium. This occurs either byesterification of the fatty acid derived from the fat hydrolysis withthe loss of water, where this latter product migrates to the aqueousphase, or by transesterification of the oils or fats with loss ofglycerol, where this latter product likewise moves into the aqueousphase. Thus, the chemical equilibrium in the organic phase liescompletely-on the side of the ester.

The addition of more than the stoichiometric amount of alcohol willshift the equilibrium further toward the ester side, and the rate ofreaction will be increased significantly. In many cases the enzymaticfat hydrolysis of the present invention is nearly complete even with theaddition of a slight excess of 5% over the stoichiometric amount ofalcohol. This has been demonstrated for a series of n- and iso-alcoholsfrom C8 to C24. The hydrophobic organic phase contains no mono-, di- ortriglycerides. It consists solely of the fatty acid esters of the addedalcohol, a small amount of the free fatty acids along with thecorresponding amount of alcohol, and the excess alcohol. It is possibleto remove the excess alcohol, including the unreacted alcohol portion,and the free fatty acids from the organic phase, in order to obtain thepure esters.

The methods used to recover the pure esters depend on the nature of thefatty acids and the alcohols. For instance, if esters of C18 fatty acidsand C18 alcohols are produced from corresponding oils or fats andalcohols, it is possible to remove the free C18 fatty acids along withthe excess C18 alcohol from the end product of thehydrolysis/esterification reaction by distillation, because the fattyacid esters produced have a substantially lower vapor pressure than thefree fatty acids and alcohols.

It is possible for the fatty acids and unreacted alcohol removed bydistillation to be recycled to the combined hydrolysis/esterificationreaction, so that they will likewise be converted to esters withoutloss. In continuous operation, then, only the stoichiometric amount ofthe starting oil is added, while the residual amounts of alcohol and thefree fatty acids are recycled.

In some cases, the free fatty acids and the resulting esters may haveapproximately the same vapor pressure. This occurs, for example, in theesters of C18 fatty acids with C 13 iso-alcohols. In these cases, it ispossible to remove the excess amounts of C13 iso-alcohol by distillationand to separate the free C18 fatty acids from the ester/fatty acidmixture in the distillation pot by base extraction, followed byneutralization, so that these are available for recycling.

One interesting variant of fat hydrolysis with an integratedesterification or transesterification occurs with use of oils in whichthe fatty acids are not randomly bound to the alcohol groups of theglycerol but rather systematically, as for example the erucic acid incrambe oil. In this case, with proper selection of the hydrolyzingenzyme, it is possible to target the production of only the erucic acidesters of the added alcohols and monoglycerides of the other fatty acidsof the crambe oil, or erucic acid diglycerides and fatty acid esters ofthe non-erucic acids. The resulting mixtures are separated by methodsknown to the art, preferably by distillation. For example, it ispossible to separate by distillation the mixture of erucic aciddiglycerides, excess C13 iso-alcohol, and C18 fatty acid—C13 iso-alcoholester obtained, after using a chain-length specific hydrolyzing enzymeand iso—C13 alcohol as the alcohol component. Particularly after theseparation of a component formed selectively by a specific enzyme (e.g.,the C18 fatty acid—C13 iso-alcohol ester), it is advantageous to subjectthe mixture of substances resulting from the enzymatichydrolysis/esterification to a subsequent, second enzymatic hydrolysisprocess using another enzyme. This method allows one to obtain the fattyacids or fatty acid esters for which this latter enzyme exhibitsselective catalytic action in the hydrolysis and esterification process.For example, it is possible to convert an erucic acid diglyceride, freeof C 18 fatty acids as explained later, to glycerol and erucic acid, orto an erucic acid ester in the presence of an alcohol, in a secondenzymatic hydrolysis process using an enzyme which is not chain-lengthspecific.

The process of enzymatic fat hydrolysis starting from oils and fats ofthe present invention thus allows the targeted production of fatty acidsand fatty acid esters which previously could be obtained only atsubstantially higher cost. This process utilizes the known action oflipases as biocatalysts that establish a chemical equilibrium betweenesters, alcohols, water and acids. They act particularly on fats andoils. The latter are glycerol esters, primarily triglycerides, ofmedium- to long-chain and generally unbranched fatty acids. Lipasesoperate at the phase boundary of two-phase systems having oil or fattyacids as the hydrophobic phase and water as the hydrophilic phase, andestablish a chemical equilibrium in both phases. The position of thechemical equilibrium is determined by the concentrations of theparticular materials in the particular phases.

In the phase system of water and oil or native fatty acid, the waterconcentration is dominant in the water phase but very slight in the oilphase. As glycerol is very highly soluble in water, but hardly solubleat all in the hydrophobic phase of oil or hydrophobic fatty acids, theglycerol concentration at equilibrium must be substantially higher inthe water phase than in the oil phase. Glycerol present or formed in theoil phase passes almost completely into the water phase. Native fattyacids with medium to long fatty acid chains are hydrophobic and almostinsoluble in water. Their concentration in the water phase isconsequently very low. On the other hand, they are quite soluble in thehydrophobic phase, and they themselves will occasionally constitute thehydrophobic phase. When lipases act on the oil at the water/oilinterface, the oil is hydrolyzed with the consumption of water todiglycerides and monoglycerides, and finally to fatty acids andglycerol. At equilibrium, the law of mass action applies to both phases:Water phase {[fatty acid]³ · [glycerol]}/{[triglyceride] · [water]³} = KOil/fatty acid phase {[fatty acid]³ · [glycerol]}/{[triglyceride] ·[water]³} = K

For the system of reaction equations for hydrolysis of triglyceride tofatty acids: 1.) Triglyceride + H₂O

diglyceride + fatty acid 2.) Diglyceride + H₂O

monoglyceride + fatty acid 3.) Monoglyceride + H₂O

glycerol + fatty acid

Then, from the law of mass action: K₁ = {[triglyceride] ·[H₂O]}/{[diglyceride] · [fatty acid]} K₂ = {[diglyceride] ·[H₂O]}/{[monoglyceride] · [fatty acid]} K₃ = {[monoglyceride] ·[H₂O]}/{[glycerol] · [fatty acid]}K = K₁· K₂· K₃ = {[triglyceride] · [H₂O]³}/{[glycerol] · [fatty acid]³}

The water concentration [H₂O] in the organic phase is low and constant.As glycerol goes predominantly into solution in the water phase, theglycerol concentration, [glycerol], in the organic phase is also low andthus quasi-constant. Thus: K · [glycerol]/[H₂O]³ = K′ =[triglyceride]/[fatty acid]³

Because of the differences in the solubilities of the individualcomponents in the hydrophilic and hydrophobic phases, lipases hydrolyzefats and oils almost completely to glycerol and fatty acids when thewater concentration in the hydrophilic phase is high. The glycerol thatis formed thus dissolves in the water, and the fatty acids initiallydissolve in oil, but later form a separate hydrophobic fatty acid phase.

If the enzymatic fat hydrolysis of the present invention is carried outin the presence of added alcohols other than the glycerol contained inthe fats and oils, the lipases will likewise produce a chemicalequilibrium in the hydrophilic and hydrophobic phases. Exceptions tothis are found for alcohols and alcohol concentrations that inhibit theactivity of the enzyme, or which are incompatible with it and inactivateit. Even in this case, the position of the chemical equilibrium isdetermined by the distribution coefficients of the individual componentsbetween the hydrophilic and hydrophobic phases. The calculation orestimation of the chemical equilibrium distribution in two phases isnaturally more complex than for simple fat or oil hydrolysis. That isparticularly true for multifunctional alcohols which are water-soluble,for example trimethylolpropane and pentaerythritol.

The behavior of hydrophobic alcohols that are practically insoluble inwater (both monohydric and polyhydric alcohols) is considerably easierto calculate. The medium-chain (starting with a C6 chain length) tolong-chain n- and iso-alcohols of the present invention are included inthis group. Such alcohols when added to a two-phase system will inpractice be soluble only in the organic phase. The glycerol-containingaqueous phase will contain very little alcohol as a consequence of itslow saturation concentration. Addition of such alcohols shifts thehydrolysis equilibrium toward hydrolysis because of esterification andconsumption of free fatty acids. This effect is increased by theaddition of excess alcohol, so that there will no longer be any tri-,di- or monoglycerides in the organic phase at equilibrium. For suchalcohols, which are quite soluble in the organic phase, the abovereaction in the presence of alcohol is described as follows:

This is essentially independent of the nature of the alcohol: Ester +H₂O

fatty acid + alcohol K_(ester) = {[ester] · [H₂O]}/{[fatty acid] ·[alcohol]}

In contrast to water-soluble alcohols, for alcohols which are quitesoluble in the organic phase: [alcohol] is saturated in H₂0, and so isconstant. Thus: K′ · K_(ester)/[H₂O] = K″ = {[triglyceride] · [ester]}/{[fatty acid]⁴ · [alcohol]}The process of the present invention is carried out very successfullywith alcohols whose water solubility is less than 5 wt.%, based on theaqueous phase. The yield and conversion rate decrease with increasingwater solubility of the alcohol. For instance, when TMP was used as thealcohol in the process of the present invention, the conversion ratedecreased by about 50%.

Some lipases, called specific lipases, are unable to hydrolyze all thefatty acid ester glyceride bonds. In particular, it is not possible forcertain lipases to hydrolyze the central fatty acid bonded to theglycerol C2. Such lipases are used to target the production ofmonoglycerides and fatty acids, for example. If the starting oilscontain certain fatty acids that are not bound in the glyceride of theoil or fat randomly, but rather are systematically distributed, then itis possible to obtain fatty acids or their esters which do notcorrespond to the fatty acid pattern present in the triglyceride thoughthe use of specifically acting lipases.

For instance, it is known that long-chain fatty acids with chainlengths >20, such as erucic acid, are always bound to the outer hydroxylgroups of glycerol in native oils and fats, and not to the centralhydroxyl group. In oils rich in erucic acid such as crambe oil, forinstance, with more than 60% by weight erucic acid (and about 6% byweight of fatty acids >C22), practically all the fatty acids havingchain lengths >C20 are bound to the glycerol C1 and C3, while theremaining 33.33 mole-percent of C18 fatty acids are bound to theglycerol C2. In this case, a specific lipase is used to target thecleavage of only the terminal acids and produce their esters. Then, theerucic acid esters are isolated by fractional crystallization, forexample. In this case, the enzyme also acts at the phase boundary layer,and it is possible for it to be recycled efficiently according to thepresent invention.

It is also known that the activities of particular lipases often dependon the chain length of the particular fatty acids or on the degree ofand the configuration (cis, trans, conjugated or not conjugated, etc.)of unsaturation of the fatty acids. The process of the present inventionalso utilizes these effects for targeted production of fatty acids ortheir esters.

In this case, too, the enzyme catalysis occurs in the boundary layerbetween the hydrophilic and hydrophobic phases. Recycling of the enzymeand removal of components enable the targeted production of the desiredproducts. This is explained with the above-mentioned example of crambeoil.

The nonspecific lipase from Candida rugosa cleaves long-chain fattyacids such as erucic acid considerably more slowly than C16 and C18fatty acids. When carried out according to the present invention, theenzymatic fat hydrolysis using Candida rugosa lipase yields the1,3-diglyceride of erucic acid along with the fatty acids cleaved fromthe glycerol C2. These latter are C 18 fatty acids, and these fattyacids and their esters are separated advantageously from the diglycerideas the distillate when short-path distillation is used. It is possibleto realize similar results for many other fatty acids such as omega-3-and omega-6-fatty acids by using suitable enzymes.

As noted above, the separation of the fatty acids from the reactionproducts of the hydrolysis reaction or, alternatively, the separation ofthe fatty acid esters from the reaction products of thehydrolysis/esterification reaction, is preferably carried out by vacuumdistillation, and especially by gentle short-path distillation.. If thefatty acid esters produced have lower vapor pressure than the free fattyacids and alcohols, then the distillate contains the excess amount of n-or iso-alcohols, the unreacted alcohol, and the free fatty acids. Thedistillate is preferably recycled back to the hydrolysis/esterificationprocess. It is possible to use adsorptive separation (such as columnchromatography) as an alternative to the separation of the fatty acidesters or fatty acids by distillation.

Vacuum thin-film evaporators such as falling film evaporators orshort-path stills are gentler than batch distillation, and above all areoperated continuously, and so their use is preferred. In any case, thesedistillation techniques also require a liquid residue of at least 5% to10%, as otherwise the distillation film will break. This condition issatisfied in the present invention for both the hydrolysis and thecombined hydrolysis/esterification processes.

As previously discussed, the residue from the distillation in thecombined hydrolysis/esterification process contains either the desiredester, or the ester and unreacted free fatty acids. In the case of areaction involving only hydrolysis, the distillate contains thehydrolyzed fatty acids. The pot, or distillation residue, contains theunreacted triglycerides, and is recycled back to the hydrolysis process.

The emulsion-like intermediate layer collected in the centrifugeaccording to the present invention, in the plate pack of a separator,according to the preferred embodiment, is discharged discontinuously bycompletely emptying the drum periodically, and the enzyme-containingintermediate layer obtained is reused. In this case it is convenientalways to empty the drum when the discharged organic phase and/or theglycerol-containing aqueous phase begins to become turbid with thedischarged intermediate layer. It is also possible to empty the drummore frequently at specified times, but this is not preferred. The factthat both the aqueous and organic phases are also discharged when thedrum is emptied fully is not disadvantageous because all the phases arereused by recycling them back into the reactor. It is also possible forthe drum to be emptied partially instead of fully, and these proceduresshould be arranged so that the intermediate layer is discharged ascompletely as possible.

The losses of enzyme through discharge with the aqueous and organicphases are reduced drastically in the manner shown. The discharge lossesin the process of the present invention are very slight compared withtime-dependent enzyme consumption (enzyme aging).

A technically interesting supplement to the invention for furtherreducing the enzyme loss in the separated organic phase consists ofusing an additional self-discharging polishing separator. Thisadditional separator is configured immediately after the separator forthe single or last hydrolysis or hydrolysis/esterification step andreceives the organic phase discharged from it. This added separator ispreferably a self-discharging centrifuge with a plate pack adjusted sothat the solids undergoing sedimentation, the enzyme in this case, andthe remnants of the aqueous phase which are still removable willseparate at the drum wall. Then the quantities of enzyme centrifuged offin this manner are discharged discontinuously again and recycled back tothe hydrolysis or hydrolysis/esterification process.

When a self-discharging centrifuge is used to separate the organic phasecontaining the fatty acid esters, it is particularly advantageous tocarry out the hydrolysis reaction in loop reactors, or in other words,intermittently or batchwise, not continuously in flow reactors. Thus,for instance, a reactor is filled with oil, so that its loop is notactive for circulating the oil or fat, water, alcohol and enzymecontained in the reactor. At the same time a second reactor carries outthe hydrolysis or hydrolysis/esterification reaction with an activeloop. Also at this time, a third reactor is being emptied through anself-discharging centrifuge, so that the reaction mixture is separatedinto the glycerol-containing aqueous phase, the organic phase containingthe separated fatty acids or fatty acid esters, and theenzyme-containing emulsion boundary phase which forms as an intermediatelayer. The emulsion boundary phase is discharged discontinuously fromthe separator from time to time, replenished with fresh enzyme, andreturned to the reactor.

It is possible to run the reaction at a high enough enzyme concentrationas to produce an unusually large phase boundary due to the circulationand the shear fields produced in the circulation pumps during the loopreactor operation. Thus it is possible to keep the amount of added waterlow and to obtain an aqueous phase with a considerably higher glycerolconcentration than was previously possible in the hydrolysis processesmentioned. In this case, there is no significant lengthening of thereaction time even with more than 30 wt.% glycerol in the dischargedaqueous phase. It has not previously been considered feasible to obtainsuch high glycerol concentrations. Furthermore, the high glycerolconcentrations will drastically reduce the loss of enzyme.

An advantage of the present invention is that the amount of added wateris low, both in the case involving only the hydrolysis process, as wellas for the combined hydrolysis/esterification process. In the lattercase, a minimum of 5% water by weight is added, based on the organicphase, including the oil or fat and alcohol used. It is possible to addmore than 200% by weight water, but that just complicates the entireprocess unnecessarily. In order to take advantage of the high glycerolconcentration in the range of 10 to 35% by weight of the resultingglycerol-containing aqueous phase made possible in the presentinvention, the amount of added water should preferably be in the rangeof 20 to 30% by weight based on the organic phase used, and not over 50%by weight. Even in the process involving only hydrolysis, one workspreferably in the range of 5 to 200% added water based on the organicphase used, preferably 20 to 30% and maximally 50% by weight.

It is possible to run the process of the present invention at a highenzyme level without consuming much enzyme. According to the presentinvention, even when the enzyme activity is high, the amount of lipaseadded to the reactor as an effective amount is generally at least 0.01wt.%, based on the oil or fat used. The present preferred range for theamount of lipase used in the working examples is between 0.1 and 0.5% byweight, based on the oil or fat used. This high enzyme level greatlyaccelerates the hydrolysis as well as the hydrolysis/esterificationprocess. In addition, the actual enzyme consumption is very low becausethe enzyme is recycled, so that only fractions of the initial amountsneed to be added later. In actual runs, the amounts of lipase addedlater were less than ten percent of the active amount placed in thereactor. One skilled in the art knows that the optimal enzyme level isselected not only for the specific enzyme, but also depends on theactivity of the particular enzyme preparation. Finally, one mustconsider that the process becomes steadily slower as the enzyme level inthe process decreases. It is also known, and has previously been statedin the publications mentioned above, that increasing the enzyme levelabove certain values gives no advantage with respect to the processtechnology or economics. It is possible for one skilled in the art todetermine the optimum enzyme level for the particular starting materialswith a few experiments by considering these facts.

According to the present invention, it is possible for the hydrolysis orhydrolysis/esterification reaction to be carried out in just one stagein the presence of alcohol with complete release of the glycerol, withrecycling of the enzyme in this stage.

However, the hydrolysis or hydrolysis/esterification reaction of thepresent invention is preferably carried out in multiple stages withcirculating reactors, for example in two stages. The aqueous glycerolphase obtained in the second stage is then recycled back to the firststage as the aqueous phase, and fresh water as the water aqueous phaseas along with the organic phase obtained from the first stage are alsocycled to the second stage. Such a two-stage or multi-stage reaction isadvantageous because the enzyme loss is further minimized.

The process of the present invention is also suitable for hydrolyzingthe mono-, di-, and triglycerides from soap stock obtained from thealkali refining of feed oils, and converting them to fatty acid esters.For this purpose it is preferable to release the fatty acids bound inthe soaps by adding acid before the hydrolysis/esterification.

The invention will now be exemplified using working examples and theaccompanying drawings, in which

FIG. 1 a shows a schematic representation of an example of an industrialprocess of the present invention, designed for the case in which theresulting fatty acid esters are to be separated from the unreacted fattyacids and alcohol by distillation;

FIG. 1 b shows a corresponding schematic representation of a modifiedprocess for the case in which the fatty acid esters are only separatedtogether with the free fatty acids by distillation, and

FIG. 2 shows a schematic representation of an example of an industrialhydrolysis process of the present invention, in which no esterificationoccurs and which is operated without addition of alcohol.

FIGS. 1 a and 1 b show a configuration of the present invention for atwo-stage combined fat hydrolysis/fatty acid ester formation which issuitable for realizing the features noted on a production scale. Twoprocess stages 1 and 2 are provided, each of which comprises threecirculation loop reactors. As already discussed above, the reactors areoperated intermittently: filling, reaction phase, emptying phase. Thecirculation loop for each of the three reactors in a stage is furnishedwith a centrifugal pump indicated in the drawing, and preceded by a heatexchanger. The reactors are stainless steel vessels, for instance, andare equipped with stirrers. Also, a separator in the form of anself-discharging plate separator is provided for each stage.

The exit port of the separator for the second hydrolysis stage, fromwhich the organic phase is discharged with the fatty acid esters, isconnected to a vacuum short-path still, in which a short-pathdistillation is effected to separate the free fatty acids and thealcohols from the fatty acid esters formed, for the case presented abovein which the latter have a lower vapor pressure than the former.

The residue from the distillation will contain the desired esters if theunreacted free fatty acids and the alcohol are more volatile than thedesired fatty acid ester end product. This corresponds to the processshown in Figure 1 a.

If, instead, only the alcohol is obtained as the distillate, as in FIG.1 b, the residue from the distillation contains both the esters and thefree fatty acids. The free fatty acids are neutralized in a separator byadding an alkaline solution such as sodium hydroxide, and then areseparated from the fatty acid esters as the heavier soap phase bycentrifugation. The soap phase is cleaved to provide fatty acids andsalts by a known method, e.g., in a second centrifuge after addition ofan acid such as sulfuric acid, and the fatty acids are returned to thefirst stage of the reaction.

The reactor is charged with a buffer solution, the triglyceride to behydrolyzed, the particular ester-forming alcohol, and the enzyme, i.e.,lipase. More enzyme is obtained in each time the separator is emptiedintermittently, and is returned to the reactor of the same stage that isbeing filled, with each particular separator routed to a particularreactor. Thus the enzyme remains in circulation in one stage, along withthe discharged proportions of free fatty acids, unhydrolyzedtriglycerides, etc. This prevents the partial mixing of startingmaterials of different quality from the two stages. This also reducesthe risk of reverse reactions. Finally, it should also be mentioned thata glycerol solution is removed from the first-stage separator as theseparated heavier liquid phase and is ready for further processing. Theglycerol solution from the second-stage separator is returned to thefirst-stage reactor that is being filled, as shown in the figures.

A slightly acidic standard solution selected and adjusted according tothe conditions specified by the enzyme producer for use as the buffersolution. An aqueous solution with sodium acetate and acetic acid,adjusted to be slightly acidic, is used in the working examples. Theoptimal pH of the buffer solution is adjusted for the particular enzyme.

This also applies for the process control temperature. Temperaturesbetween 25° C. and 45° C. have been tested in experiments. Here itshould be noted that the temperature is slightly elevated in generalbecause of the exothermic reaction. In the cases tested, though, it waseasily possible to ensure that the aqueous and organic phases were keptliquid and that no crystallization occurred in the liquid phases.

In principle, it is possible for any type of enzyme presented in theabove-mentioned Bühler and Wandery publications to be used. VariousCandida rugosa enzymes were tested thoroughly. It is also suitable touse enzymes obtained from oil seeds in the process of the presentinvention (e.g., from castor oil). They will naturally have theadvantage of a particularly selective activity for certain fatty acids.There has been scant use of such enzymes for fat hydrolysis, however,and they are generally more costly than other enzymes prepared byindustrial fermentation of yeasts, molds, bacteria and the like.

Oleyl alcohol and stearyl alcohol were tested as the n-alcohols in theexperimental series. Experimental series were carried out with iso-C8,iso-C10, iso-C13, iso-C16, iso-C18, iso-C20 and iso-C24 as iso-alcohols.

The esters were prepared from high-oleic sunflower oil. Depending on theapplication, it is possible to start with dewaxed and refined, or crudeoil or fat.

It is also possible to carry out the invention with less available andcertain longer-chain alcohols, from greater than C26 up to C36, and withother oils and fats.

EXAMPLES

1. Enzymatic Esterification

An 80 liter stirrer vessel was charged with 20 kg of dewaxed and refinedhigh-oleic sunflower oil 90plus®, and 22.3 kg (10% stoichiometricexcess) of Isofol 20 (C20 alcohol from Fuchs Petrolub), 10.6 kg buffersolution (0.1 N sodium acetate/acetic acid, pH 4.6) and 40 g of OFEnzyme 360 (from the Meito Sangyo company) was blended in. This mixturewas circulated with a centrifugal pump for 3 hours at about 40° C.

Then the combined phase mixture was moved directly by gravity feed at arate of about 30 kg/hr to a plate centrifuge (SA 1-01, WestfaliaSeparator AG, Oelde) and separated continuously. The acid value of thedischarged organic phase, as determined by titration with 0.1 N KOH inalcoholic solution, according to DIN 53169 and DIN 53402, passed througha maximum of about 55, and decreased to about 15 at the end.

The enzyme was discharged from the centrifuge together with smallamounts of the organic phase and glycerol-water. It showed hardly anyloss of activity and could be reused.

A clear oil-ester phase and a clear glycerol solution as the aqueousphase were taken from the centrifuge. The aqueous phase contained 17% byweight of glycerol as expected.

No tri-, di- or monoglyceride could be detected in the oil-ester phaseby thin-layer chromatography. After a final vacuum distillation of a 4kg aliquot, a residue of the corresponding C20 iso-ester was obtained in95% yield, based on the amount of oil used.

The distillate contained the excess amount and the residual unreactedportions of iso-alcohol and free fatty acids.

In a laboratory experiment, a stoichiometric mixture of oil, iso-alcoholand buffer solution was added to part of the distillate, so that thealcohol excess, based on the new amount of oil used, was again 10%.

The yield likewise reached 95% over the same time and with the sameamount of enzyme. Here, too, comparable quantities of unreactediso-alcohol and free fatty acids could be distilled out of the organicphase that was centrifuged off after termination of the reaction, sothat it was possible to circulate the distillate without loss. It wasalso shown that the enzyme used (OF 360) catalyzes the esterification offree fatty acids and iso-alcohol in the presence of an aqueous phase.

The effectiveness of the process of the present invention was furthertested for the n-alcohols oleyl alcohol and stearyl alcohol. Forexample, an experiment was carried out with equivalent success usingoleyl alcohol (MW =268.49) and the lipase used in the example above.Iso-C8, iso-C10, iso-C13, iso-C16, iso-C18, iso-C20 and iso-C24 wereused as iso-alcohols. Experiments were also done successfully withcrambe oil, whereby the other possibilities presented above weredemonstrated. A mixture of branched C16/C18 fatty alcohols (MW =286) wasalso converted successfully with the lipase above according to thepresent invention.

In principle the process of the present invention is also applicable tosynthetic fatty acid esters, such as synthetic triglycerides and otherpolyol esters.

2. Enzymatic Hydrolysis

FIG. 2 shows clearly that the process control for the process involvingonly fat hydrolysis without simultaneous esterification by addition ofalcohol does not differ in the essential points from the foregoing, forexample the process control presented above in Figures 1 a and 1 b.Possible mass throughputs are reported for examples, but the process canbe and has been also carried out successfully with other quantities.Therefore only the distinguishing part of the process is explained. Thestatements above about possible temperature ranges and handling of theenzyme apply equally.

Thus the exit port of the second hydrolysis stage separator, from whichthe organic phase with the fatty acids (instead of the fatty acid ester)is discharged, is again connected to a vacuum short-path distillationsystem in which short-path distillation is used to separate the freefatty acids. The residue from the distillation was sent to a stirredcrystallizer in which the waxes crystallized. In connection with that,the residual oil in which the waxes and other higher-boiling componentshad crystallized out as solids was pumped into a filter assembly, whereit was freed of those concomitants. The oil purified in that manner wasthen returned to the first stage for hydrolysis.

A hydrolysis of high-oleic sunflower oil was carried out. After astarting phase, 30.0 kg of a crude, unrefined high-oleic sunflower oil90 plus (registered trademark) from the Dr. Frische GmbH company, havingan acid value of 4 as determined by titration with alkaline potassiumhydroxide according to DIN 53169 and DIN 53402, was charged to one ofthe first-stage reactors along with 7.0 kg of a buffer solutionconsisting of a 12% glycerol/water solution buffered with 3.0 g sodiumacetate. The charged mixture of oil and buffer solution was circulatedby means of a centrifugal pump with stirring and maintained at 35 - 40°C. Next, 2 kg of enzyme-enriched discharge product from the firstseparator stage of the start-up phase was added, along with 3 g freshlipase from Candida rugosa (lipase in the form of a powdered solid fromMeito Sangyo, Japan, 360,000 units/gram) which had also been used in thestart-up phase. After 60 minutes reaction time with stirring and thenbeing allowed to settle, the reaction mixture was separated in the firststage separator (self-discharging plate centrifuge SA1-01 from WestfaliaSeparator AG Co.) into an organic phase containing fatty acids and anaqueous glycerol phase. The reactor content was fed to the separator at40 kg/hour, and the centrifuge drum was completely emptied hydraulicallyevery 15 minutes. The discharge product from the drum was transferred tothe particular first-stage reactor that was being filled. Thesecond-stage reactor to be filled received, along with the oleicacid-containing organic phase from the first stage separator, 7.0 kg ofthe above-described buffer solution, 2 kg of the discharge product fromthe second-stage separator from the start-up phase, and replenished with5 g fresh lipase of the type noted above. Otherwise, this process wascarried out as in the first-stage fat hydrolysis, with the result thatthe lighter phase discharged from the second-stage separator was a clearcrude oleic acid phase with an acid value of 184, corresponding to a 93%conversion for the hydrolysis (calculated as the measured acid valuedivided by the theoretical acid value for this mixture). The heavy phaseseparated by the centrifuge constituted a 12% by weight solution ofglycerol in aqueous buffer solution. The crude fatty acid thus obtainedin the second stage was collected in a 200 liter vessel serving as anintermediate container. This was subjected to short-path distillation ina short-path vacuum still, Type KD, from UIC Co., Alzenau-Hörstein, withan initial degassing stage to separate any traces of water. For example,in a distillation at 191° C. and system pressure of 0.014 mbar, about 8%by weight of the fatty acid used came out continuously under theseconditions as a residue of non-boiling components. The other 92% byweight of the crude product distilled as oleic acid having an acid valueof 199-200, with only the slightest amounts of minor constituents in theform of fatty acids with a lower vapor pressure. This was consistentwith the theoretically calculated acid value, within the accuracy of themeasurement. The value was somewhat higher than the theoretical acidvalue because of the small proportion of more volatile, shorter fattyacids.

The residual product was dewaxed, in which the higher fatty acids (chainlengths of C22 and higher), waxes, and other higher-boiling componentscontained in the oil crystallized out as solids and were separated byfiltration. The filtered residual product was returned to the hydrolysisprocess at the first stage by a pump as indicated in FIG. 1.

Some other examples were run in the same manner, including some withhigher proportions of enzyme and correspondingly shorter hydrolysistimes; with beef tallow, and with crambe oil. In the example with crambeoil, which contains 60% erucic acid, a mixture of the non-specificenzyme OF 360 noted above and the 1,3-specific enzyme Novozym 388 wasused. The latter specifically hydrolyzes off the fatty acids bound tothe 1- and 3-positions of the triglyceride structure, erucic acid inthis case.

1. A process for the enzymatic hydrolysis of oils and/or fats withsimultaneous enzymatic formation of fatty acid esters using lipasesacting as biocatalysts and alcohols, especially n- and iso-alcohols,said process comprising: causing lipases, as biocatalysts for hydrolysisof oil or fat and formation of fatty acid esters in a fathydrolysis/esterification, to act on a mixture of triglycerides, water,and an alcohol soluble in oil or fat to create a reaction mixture formedin the fat hydrolysis/esterification; transferring the reaction mixtureto a drum of a self-discharging centrifuge for separation into aglycerol-containing aqueous phase and an organic phase that contains thefatty acid esters which have formed; adjusting the centrifuge so that alipase-enriched intermediate layer formed in the centrifuge between theaqueous phase that is drained off and the organic phase that is drainedoff is accumulated in the centrifuge; and emptying contents of the drumof the centrifuge, the drum contents including the accumulatedintermediate layer, at specified times for recycling to the combinedhydrolysis and esterification process or are made ready for a furtherhydrolysis and esterification process.
 2. The process of claim 1,wherein the alcohol is used in an excess of 2 to 100%, preferably 5% to20%, based on the stoichiometric amount required for esterification. 3.The process of claim 1, wherein the amount of water added is at least 5%by weight based upon the organic phase employed that is comprised of oilor fat and alcohol.
 4. The process of claim 1, wherein the alcohol usedis an alcohol which is quite soluble in the organic phase formed, but isconsiderably less soluble in water, and especially medium-chain tolong-chain n- and iso-alcohols.
 5. The process o f one of claim 1,wherein the fat hydrolysis/esterification is carried out discontinuouslyin reactors that are run in loop operation with the reactor contentscirculated by pumps, with multiple reactors being provided in parallelfor a single, or for each added, reaction stage, with one of thereactors being filled with the circulating loops not active, with ahydrolysis/esterification operation being run in a second reactor havingactive circulating loops, with a third reactor having its circulatingloops not active and being emptied through a centrifuge which separatesthe glycerol-containing aqueous phase formed in the hydrolysis processfrom the organic phase containing the fatty acid esters prior to whenthe fatty acid esters are separated from the organic phase.
 6. Theprocess of claim 1, wherein the lipases is selected from a groupconsisting of non-specific lipases, specific lipases, or mixtures ofnon-specific and specific lipases.
 7. The process of claim 1, whereinthe free fatty acids and alcohol from the organic phase are separated bydistillation from the fatty acid esters that have formed and theseseparated free fatty acids and alcohol are returned to thehydrolysis/esterification process.
 8. The process of claim 1, whereinthe organic phase drained out from the self-discharging centrifuge istransferred to another self-discharging centrifuge, which is likewiseemptied intermittently to recover lipase residues that have collected asa sediment on the centrifuge wall for reuse in the hydrolysis process.9. A device for carrying out the process of the enzymatic hydrolysis ofoils and/or fats with simultaneous enzymatic formation of fatty acidesters using lipases acting as biocatalysts and alcohols, especially n-and iso-alcoholsone, said device comprising: at least onehydrolysis/esterification reactor for use in a hydrolysis andesterification process; at least one self-discharging centrifuges inwhich a lipase-enriched phase formed in the at least onehydrolysis/esterification reactor between an aqueous phase that isdrained off and an organic phase that is drained off, accumulates in adrum of the at least one centrifuge which is emptied at specified times;a feedback system for returning contents of the intermittently emptieddrum including said lipase-enriched phase from the at least onecentrifuge to the combined hydrolysis and esterification process; and aa means for separating alcohol, free fatty acids and fatty acid estersformed from the organic phase that is supplied from the at least onecentrifuge.
 10. The device of claim 9, wherein the means for separationis a distillation apparatus.
 11. A process for the enzymatic hydrolysisof oils and/or fats using lipases acting as biocatalysts to obtain fattyacids and glycerol, said process comprising: causing lipases to act asbiocatalysts on a mixture of an oil or fat and water to hydrolyze theoil or fat to produce a reaction mixtures; transferring the reactionmixture thus produced to a self-emptying centrifuge for separation intoa glycerol-containing aqueous phase and an organic phase that containsfree fatty acids that have been hydrolyzed off in the precedinghydrolysis; adjusting the centrifuge so as to accumulate alipase-enriched intermediate phase that forms in the centrifuge betweenthe aqueous phase that is drained off and organic phase that is drainedoff; and emptying the centrifuge of drum contents at specified times andthe centrifuge drum contents that have been emptied from the centrifugeare returned to the hydrolysis process or are prepared for a furtherhydrolysis process.
 12. The process of claim 11, wherein the fathydrolysis is carried out discontinuously in reactors that are run inloop operation with the reactor contents circulated by pumps, withmultiple reactors provided in parallel for a single or for each ofnumerous reaction stages, with one of the reactors being filled with thecirculation loops not active, with a hydrolysis operation being run in asecond reactor having active circulation loops, with a third reactorhaving its circulating loops not active and being emptied through acentrifuge which separates the glycerol-containing aqueous phase formedin the hydrolysis process from the organic phase containing the freefatty acids prior to when the fatty acids are separated from the organicphase.
 13. The process of claim 11, wherein the free fatty acids andalcohol are separated by distillation out of the organic phase from thefatty acid esters that have formed, and are returned to thehydrolysis/esterification process.
 14. The process of claim 11, whereinthe organic phase flowing out of the self-discharging centrifuge istransferred to another self-discharging centrifuge, which is likewiseemptied intermittently to recover residues of lipase that have collectedas a sediment on the centrifuge wall for reuse in the hydrolysisprocess.
 15. A device for carrying out the process for the enzymatichydrolysis of oils and/or fats using lipases acting as biocatalysts toobtain fatty acids and glycerolany, said device comprising: at least onehydrolysis reactor for use in a hydrolysis process to form a reactionmixture; at least one self-discharging centrifuges in which the reactionmixture is separated into an aqueous phase that is drained off and anorganic phase that is drained off, wherein a lipase-enrichedintermediate layer being formed between said aqueous phase and organicphase is accumulated in the centrifuge, and which is emptied atspecified times; a feedback system for transporting contents of theintermittently emptied at least one centrifuge including theintermediate layer from the centrifuge to the hydrolysis process; and ameans for separating free fatty acids from the organic phase that issupplied from the centrifuge.
 16. The device of claim 15, wherein themeans for separation is a distillation apparatus.
 17. The device ofclaim 16, wherein said distillation apparatus is selected from a groupconsisting of a short-path still and a falling film evaporator.
 18. Thedevice of claim 10, wherein said distillation apparatus is selected froma group consisting of a short-path still and a falling film evaporator.19. The process of claim 14, wherein the another self-dischargingcentrifuge is a polishing centrifuge.
 20. The process of claim 8,wherein the another self-discharging centrifuge is a polishingcentrifuge.